MiR-125 inhibited cervical cancer progression by regulating VEGF and PI3K/AKT signaling pathway.
ABSTRACT: BACKGROUND:MiR-125 has been shown to be involved in a variety of cancers, including cervical cancer (CC). Here, our goal was to explore miR-125 functional role and molecular mechanism in cervical cancer development and progression. METHODS:qRT-PCR was employ to detect miR-125 and VEGF mRNA expression. Western blot was applied for testing protein levels (VEGF, E-cadherin, N-cadherin, vimentin, AKT, p-AKT, PI3K, and p-PI3K). MTT and transwell assays were used for detecting cervical cancer cell progression, including cell viability, migration, and invasion. RESULTS:We observed that miR-125 was downregulated, whereas VEGF was upregulated in cervical cancer tissues and cell lines (CaSki and SiHa). MiR-125 inhibited the proliferation, invasion, and migration by targeting VEGF in cervical cancer. Moreover, miR-125 negatively regulated VEGF expression in cervical cancer tissues. Finally, we demonstrated that miR-520d-5p inhibited the activation of PI3K/AKT signaling pathway. CONCLUSION:In conclusion, the findings demonstrated that miR-125 inhibited cervical cancer progression and development by suppression VEGF and PI3K/AKT signaling pathway.
Project description:We previously reported that relative to normal cervical mucus, microRNA 126?3p (miR?126?3p) is present in significantly greater amounts in the cervical mucus of patients with overt cervical cancer or precursor lesions. Here, we investigated the effects of enforced miR?126?3p expression in the cervical cancer cell line, HeLa, on proliferation, migration, invasion, apoptosis and protein expression. We transfected HeLa cells with miR?126?3p miRNA and found that proliferation, migration and invasion by cell counting, wound healing, cell migration and invasion assay were significantly reduced in these cells relative to those transfected with a negative control mimic. The levels of phosphoinositide 3 kinase (PI3K), phosphorylated 3?phosphoinositide?dependent protein kinase?1 (p?PDK1) and p?AKT proteins were lower in the miR?126?3p?transfected cells. Phosphorylated 70S6K (p?p70S6K), phosphorylated glycogen synthase kinase 3? (p?GSK3?), phosphorylated S6K (p?S6K), cyclin D1, phosphorylated p21?activated kinase 1 (p?PAK1), Rho associated coiled?coil containing protein kinase 1 (ROCK1), myotonic dystrophy?related CDC42?binding kinases ? (MRCK?) and phospholipase C ?1 (p?PLC?1) were also downregulated. This suggests that downstream effectors of the PI3K/PDK1/AKT pathway are targets for inhibition by miR?126?3p. In contrast, apoptotic?related proteins including the BCL?2?associated agonist of cell death (Bad), B?cell lymphoma?extra?large (Bcl?xL) and BCL?2?associated X (Bax), were all upregulated by miR?126?3p, resulting in increased caspase 3/7 activity and apoptosis. Thus, enforced expression of miR?126?3p inhibited cell migration and invasion and also induced apoptosis by regulating the PI3K/PDK1/AKT pathway in HeLa cells. Hence, high levels of miR?126?3p may inhibit cervical carcinogenesis, and targeting the PI3K/PDK1/AKT pathway via miR?126?3p could represent a new approach for treating patients with cervical cancer.
Project description:MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.
Project description:Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.
Project description:BACKGROUND:Zoledronic acid is the most potent osteoclast inhibitor and is widely used for advanced cancer patients with bone metastasis, but its role on cancer stem cells (CSCs) remains unclear. In the present study, we aimed to identify the stemness phenotypic characteristics of CSCs derived from cervical cancer cells and explore the anti-cancer efficiency of zoledronic acid on these cells, as well as the possible molecular mechanisms. METHODS:Stemness phenotypic identification of cervical cancer cells derived CSCs was performed via sphere formation efficiency (SFE), tumorigenesis, immunofluorescence staining, Transwell assay, and western blot. Anti-cancer efficiency of zoledronic acid on these cells (including proliferation, stemness phenotype, apoptosis, and cell cycle) was carried out through MTT assay, SFE, transwell, DAPI staining, flow cytometry, immunofluorescence, TUNEL staining, and western blot, both in vitro and in vivo. RESULTS:Enhanced self-renewal ability, including SFE and tumorigenesis, was verified in cervical cancer cells derived CSCs compared to parental cervical cancer cells. Specifically, the expression of ALDH1, Sox2, CD49f, Nanog, and Oct4 was significantly up-regulated in cervical cancer cells derived CSCs. Furthermore, enhanced migratory ability was observed in these cells along with up-regulated N-cadherin and Vimentin and down-regulated E-cadherin. Zoledronic acid inhibited cervical cancer cells derived CSCs proliferation in vitro and in vivo. The stemness phenotype of these CSCs including tumor sphere formation, migration, as well as the expression of the aforementioned associated markers was also suppressed. In addition, zoledronic acid significantly induced apoptosis and cell cycle arrest of cervical cancer cells derived CSCs in a dose-dependent manner. Mechanistically, the expression of phosphorylated Erk1/2 and Akt was significantly increased in cervical cancer cells derived CSCs compared to parental cervical cancer cells. Zoledronic acid inhibited phosphorylated Erk1/2 and Akt in cervical cancer cells derived CSCs. IGF-1, a potent stimulator for Erk1/2 and PI3K/Akt, attenuated the aforementioned anti-cancer effect of zoledronic acid. CONCLUSIONS:Zoledronic acid inhibited the growth of cervical cancer cells derived CSCs through attenuating their stemness phenotype, inducing apoptosis, and arresting cell cycle. The suppression of phosphorylated Erk1/2 and Akt was involved in this process.
Project description:Chondrosarcoma is the second most common primary malignant bone cancer, with potential for local invasion and distant metastasis. Chemokine CCL5 (formerly RANTES) of the CC-chemokine family plays a crucial role in metastasis. Angiogenesis is essential for the cancer metastasis. However, correlation of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is still unknown. CCL5-mediated VEGF expression was assessed by qPCR, ELISA, and Western blotting. CCL5-induced angiogenesis was examined by migration and tube formation in endothelial progenitor cells in vitro. CCL5 increased VEGF expression and also promoted chondrosarcoma conditional medium-mediated angiogenesis in vitro and in vivo. Stimulation of chondrosarcoma with CCL5 augmented PI3K and Akt phosphorylation, while PI3K and Akt inhibitor or siRNA abolished CCL5-induced VEGF expression and angiogenesis. We also demonstrated CCL5 inhibiting miR-200b expression and miR-200b mimic reversing the CCL5-enhanced VEGF expression and angiogenesis. Moreover, in chondrosarcoma patients showed the positive correlation between CCL5 and VEGF; negative correlation between CCL5 and miR-200b. Taken together, results demonstrate CCL5 promoting VEGF-dependent angiogenesis in human chondrosarcoma cells by down-regulating miR-200b through PI3K/Akt signaling pathway.
Project description:<b>Background</b>: Transmembrane-4-L-six-family-1 (TM4SF1) functions to regulate cell growth and mobility and TM4SF1 expression was upregulated in pancreatic cancer. This study further investigated the role of TM4SF1 in regulating pancreatic cancer epithelial-mesenchymal transition (EMT) and angiogenesis and the underlying molecular events.<b>Methods</b>: Tissue specimens were collected from 90 pancreatic cancer patients for immunohistochemical and qRT-PCR analysis of miR-141 and TM4SF1 levels, respectively. Pancreatic cancer cell lines were used for in vitro assays, while nude mice were used for the in vivo assay.<b>Results</b>: TM4SF1 expression was upregulated, whereas miR-141 expression was lost in pancreatic cancer tissues, both of which was associated with advanced clinicopathological features and poor survival of pancreatic cancer patients. Furthermore, miR-141 was able to target and reduce TM4SF1 expression in pancreatic cancer cells and miR-141 expression inhibited pancreatic cancer cell EMT <i>in vitro</i> and Matrigel plug angiogenesis and lung metastasis in nude mice. At the gene level, miR-141 directly targeted and reduced TM4SF1 expression and in turn induced E-cadherin expression and reduced VEGF-A expression by suppressing activation of the AKT signaling pathway.<b>Conclusions</b>: This study demonstrated that upregulated TM4SF1 and lost miR-141 expression were associated with advanced clinicopathological features and poor survival of pancreatic cancer patients. Lost miR-141 expression but induced TM4SF1 expression altered expression of VEGF-A and E-cadherin and promoted pancreatic cancer cell EMT and angiogenesis <i>via</i> the AKT signaling pathway, suggesting that targeting of miR-141 and TM4SF1 may be a potential therapeutic strategy to control pancreatic cancer.
Project description:The epithelial-mesenchymal transition (EMT) and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF)-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs), we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF) expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways.
Project description:PURPOSE:Persistent infection with high-risk human papillomavirus (HR-HPV) is thought to play a prominent role in the initiation and progression of almost all cases of cervical cancer. Previously, we and others found that microRNA 34a (miR-34a) may be regulated by HR-HPV E6 to contribute to the development of cervical cancer. Here, we aimed to identify the oncogenic potential and clinical significance of a known miR-34a target, WNT1, in cervical squamous cell carcinoma (SCC) development and to investigate the associated mechanisms underlying cervical SCC cell proliferation and invasion. METHODS:WNT1 and miR-34a expression levels were assessed in primary cervical lesions using immunohistochemistry and qRT-PCR, respectively. The cellular effects and the expression of its associated genes were examined in cervical SCC-derived Siha and Caski cells after siRNA-WNT1 (downregulation) or miR-34a mimic (upregulation) treatment. A cervical SCC xenograft mouse model was used to investigate the in vivo effects of miR-34a overexpression. HPV-16 E6/E7 expression was inhibited by gene promoter siRNA targeting, after which the levels of miR-34a and WNT1 were examined. RESULTS:WNT1 protein upregulation was found to be associated with a poor prognosis in cervical SCC patients. In vitro assays in Siha and Caski cells revealed that WNT1 downregulation decreased cell proliferation and invasion, inhibited WNT/?-catenin activation and affected the expression of E-cadherin and P-cadherin. MiR-34a upregulation resulted in decreased WNT1 expression. An inverse correlation between miR-34a and WNT1 expression was also observed in primary cervical SCC tissues. In addition, we found that MiR-34a could regulate an E-cadherin to P-cadherin switch (E-P cadherin switch) to inhibit cell proliferation and tumorigenesis in vitro and in vivo via inactivation of the WNT1/?-catenin pathway. Finally, we found that decreased HPV-16 E6/E7 expression resulted in miR-34a upregulation and WNT1 downregulation in Siha and Caski cells. CONCLUSIONS:From our results we conclude that WNT1, as a target of miR-34a, can promote cervical SCC cell proliferation and invasion by induction of an E-P cadherin switch via the WNT1/?-catenin pathway. Our results may provide new options for the treatment of patients with cervical SCC.
Project description:The present study aimed to evaluate the underlying mechanism of microRNA-203 (miR-203) in renal cell carcinoma (RCC) involving the PI3K/AKT signaling pathway. The results revealed downregulated miR-203 and upregulated CAV1 in RCC tissues. Upregulated miR-203 and downregulated CAV1 increased E-cadherin expression and cell apoptosis, decreased ?-catenin and N-cadherin expression and cell proliferation, migration and invasion, and blocked cell cycle entry. CAV1, a target gene of miR-203, decreased by up-regulated miR-203, and silencing CAV1 led to the inactivation of PI3K/AKT signaling pathway. In conclusion, our findings suggested that miR-203-mediated direct suppression of CAV1 inhibits EMT of RCC cells via inactivation of the PI3K/AKT signaling pathway.
Project description:The present study aimed to investigate the mechanism by which cyclooxygenase?2 (COX?2) promotes the metastasis of MG?63 osteosarcoma cells through the PI3K/AKT/NF??B pathway. To achieve this, a recombinant lentivirus containing the COX?2 gene was constructed in order to overexpress COX?2; a recombinant lentivirus containing a control sequence was also constructed. A Transwell chamber migration assay was performed to quantify the migration of the COX?2?transduced cells, and of cells treated with a COX?2 inhibitor (NS398) or a PI3K inhibitor (LY294002). Immunofluorescence assays were performed to determine changes in E?cadherin, vimentin and NF??B expression levels. ELISAs were performed to quantify the levels of matrix metallopeptidase (MMP)?2, MMP?9 and vascular endothelial growth factor (VEGF) in the culture medium. Western blot analysis was conducted to measure the protein expression levels of MMP?2, MMP?9, PI3K, phosphorylated (p?) PI3K, AKT, p?AKT, inhibitor of NF??? kinase (IKK) and p?IKK. The results demonstrated that the migration ability of the COX?2?overexpressing MG?63 cells was significantly increased compared with the control cells. The migration ability of cells treated with NS398 or LY294002 was significantly decreased. Compared with the control cells, E?cadherin expression was significantly decreased in COX?2?overexpressing cells, while the expression levels of vimentin, MMP?2, MMP?9, VEGF, p?PI3K, p?AKT and p?IKK were significantly increased. Compared with the control cells, E?cadherin expression was significantly increased in cells treated with NS398 or LY294002, while the expression levels of vimentin, MMP?2, MMP?9, VEGF, p?PI3K, p?AKT, and p?IKK were significantly decreased. The total protein levels of PI3K, AKT and IKK were not changed among the treatment groups. In summary, COX?2 overexpression decreased the expression levels of the epithelial protein E?cadherin and increased the expression levels of the mesenchymal proteins vimentin, MMP?2 and MMP?9, as well as promoted cell migration, by activating the PI3K/AKT/NF??B signaling pathway.